This invention relates to heterofunctionalized star-shaped poly(ethylene glycols) (PEGs) and a methods of making and using thereof. More particularly, the invention relates to star-shaped PEGs having an amine, carboxyl, aldehyde, thiol, halogen, or epoxide group on an end thereof, which allows reaction with proteins or other molecules.
Recently, much attention has been paid to peptides and proteins as drugs due to the development of recombinant DNA technology and the corresponding ease of making such peptides and proteins. Cytokines and biological response modifiers, thrombolytics, adhesion molecules, and agonist and antagonist peptide fragments of growth factors, all have wide-spread applications. From the pharmaceutical point of view, however, these drugs have several limitations such as short half-life, low stability to proteolytic digestion, and immunological side effects ranging from mild allergic reactions to anaphylactic shock. Chemical modification of a protein drug by a polymer is one of the current strategies to diminish these limitations. Poly(ethylene glycol) has been most extensively used as chemical modifying agent due to its non-toxic, non-immunogenic, and amphipathic properties. PEG has been approved by the FDA for internal use. Over 40 proteins have now been modified with PEG, including asparaginase, glutaminase, uricase, superoxide dismutase, lactoferrin, streptokinase, plasmin-streptokinase complex, adenosine deaminase, interleukin-2, N. V. Katre et al., 84 Proc. Nat'l Acad. Sci. USA 1487 (1987); catalase, arginase, insulin, b-glucuronidase, trypsin, chymotrypsin, J. Kopecek et al., 4 Bioconjugate Chem. 290 (1993); hemoglobin, recombinant human granulocyte colony stimulating factor (rhG-CSF), and recombinant human growth hormone (rhGH), R. Clark et al., 271 J. Biol. Chem. 21969-21977 (1996). Presently, asparaginase modified with monomethoxy PEG is approved by the FDA for use in patients with immunological reaction to the free enzyme.
It is well known that chemical modification by PEG almost invariably achieves increased biological half-life, reduced antigenicity, and increased resistance to proteolysis. The increased half-life is the most prominent effect of PEG modification, and is explained by several mechanisms, such as the increased size of proteins; interference with interaction of carbohydrate chains with their specific receptors; masking specific sequences for which there are cellular receptors; and reduced proteolysis and antigenicity. The extension of half-life is generally proportional to the number of PEG molecules attached per molecule. The specific activity of the protein generally decreases, however, as the degree of modification increases. The reduction of immunogenicity by PEG modification is clinically significant because life-threatening allergic reaction can be avoided. The mechanism may be via shielding of antigenic determinants by PEG, which is known to be immunologically inert. The PEG modification also protects proteins from attack by proteases and inhibitors. In addition to pharmacokinetic and immunological improvements, it has been reported that solubility in both water and organic solvents and thermal-mechanical stability increase as a result of PEG modification.
The vast majority of PEG-modified proteins, however, show some decrease in bioactivity and undergo denaturation, resulting in deactivation by chemical modification. Therefore, when a protein is chemically modified by PEG, one should carefully select the reaction conditions, as well as the configuration of the PEG molecule, so as to maximize the modification effect and minimize the protein deactivation. Due to well-established advantages of PEG modification, coupling technology regarding reaction conditions and molecular weights of PEG has been studied and reported in the literature. J. M. Harris, Poly(ethylene glycol) Chemistry: Biotechnical Biomedical Applications (1992); Y. Inada et al., 242 Meth. Enzymology 65-90 (1994); 17 Biotechnol. Appl. Biochem. 115-130 (1993); K. Yoshinga et al., 4 Bioactive Biocompatible Polymer 17-24 (1989). However, most studies to date are restricted to the modification using linear PEGs. More recently, new types of PEGs have been explored for protein modification. One-, two-, and three-branched PEG derivatives, each having only one carboxyl group in a molecule, have been obtained by reacting monomethoxy linear PEG with bromoacetic acid, protocatechnic acid, and gallic acid, respectively. I. Fuke, et al., 30 J. Controlled Release 27-34 (1994). It was found that trypsin modified with a branched PEG was greatly protected from pepsin digestion, with the degree of protection corresponding directly to the number of branches. Asparaginase modified with a two-branched PEG was obtained by reaction of monomethoxy PEG 5000 with two chlorines of trichlorotriazine. It has been shown that modification of asparaginase by a branched PEG resulted in an increase of in vivo activity, proteolytic resistance, stability, and half-life. However, these branched PEGs have been prepared by a coupling reaction using linear PEGs and multifunctional core compounds, which is cumbersome and difficult for obtaining branched PEG in high yields and purity. In addition, such a coupling process does not yield branched PEGs with a higher number of arms. Another disadvantage is that branched PEGs obtained from the coupling process have reactive functional groups interior of the PEG molecule, rendering the conjugation between the branched PEG and a protein less efficient than would be desired. Comb-shaped polyethylene oxide (PEO) has also been prepared and conjugated with bovine serum albumin and asparaginase for reducing immunogenicity. H. Sasaki et al., 197 Biochem. Biophys. Res. Chem. 287-291 (1993); Y. Kodera et al., 5 Bioconjugate Chem. 283-286 (1994).
The present invention relates to a method for making a star-shaped PEG. Star-shaped polymers comprise several linear chains linked together at one end of each chain, constituting the simplest form of branching. There exist two distinct synthetic approaches for star-shaped polymers: divergent and convergent approaches. The convergent approach, called the "arm-first method," involves the termination of growing polymer chains with multifunctional terminating agents to form the star-shaped polymer. The convergent method combined with anionic living polymerization, a newly developed technique, is known to produce a star-shaped polymer of controlled arm length, narrow molecular weight distribution, and easily varied arm number. The most common method for the synthesis of this type of polymer has involved homogeneous organolithium polymerization, followed by a linking reaction between the lithium chain end and the linking agents, such as chlorosilanes, phthalate esters, and m- and p-divinyl benzenes. The main drawback of this method is that the branches of star-shaped molecules cannot be modified with functional groups at their outer ends.
The divergent approach, also called the "core-first method," starts the synthesis reaction from a plurifunctional initiator and proceeds outward. This technique allows the modification of the branches with functional groups at their outer ends, thus providing the possibility of further reaction for forming block copolymers or selective adsorption. A problem associated with this method is the poor solubility of plurifunctional organometallic initiators, even in polar solvents. Due to its simplicity, the divergent method has been commonly applied for a variety of star-shaped gellation can happen due to a crosslinking reaction because proteins, in general, have several accessible amino groups.
In view of the foregoing, it will be appreciated that providing a heterofunctionalized star-shaped PEG and method of making thereof would be a significant advancement in the art.